Project Summary The main barriers to functional recovery after spinal cord injury (SCI) are a growth-inhibiting environment and a diminished intrinsic regenerative potential of adult neurons. Current strategies of tackling either extrinsic or intrinsic hurdles enable only limited axonal regeneration. It thus remains critical to gain novel insights into the regulatory mechanisms of glial and neuronal responses after injury. Epigenetic regulation through covalent modification of histones and DNA has emerged as a key player in glial/neuronal plasticity, however, the molecular identity, genomic targets and mechanisms of action of chromatin regulators in SCI remain unclear. At the injury site, a pro-inflammatory (M1) microglia/macrophage response predominates. Reversing this bias towards an anti-inflammatory, pro-repair M2 phenotype is predicted to facilitate tissue repair. Our preliminary data demonstrated that histone deacetylase 3 (HDAC3) is induced in M1 polarized macrophages after SCI, and that its inhibition results in increased M2 polarization and improved locomotor recovery. In Aim 1, we will investigate the basis of the neuroprotective effects of HDAC3 inhibition, focusing on M1/M2 macrophage polarization, cytokine profiles, tissue repair, and locomotor functional recovery. The study will establish HDAC3 as a key epigenetic regulator of the M1/M2 inflammatory gene network in SCI. Another hurdle for SCI recovery is the diminished intrinsic axon growth potential of adult neurons. Our recent studies established a link between histone acetylation and axon growth potential. Importantly, pharmacological inhibition of HDACs was able to reshape the chromatin landscape in mature neurons after SCI, leading to induction of regeneration-associated genes (RAG) and enhancement of axon regeneration. However, which HDAC isoform is the principal regulator of histone acetylation and RAG expression is not known. In Aim 2, we will adopt cutting-edge cell-type specific methods to examine if selective HDAC1 ablation in DRG sensory neurons would promote RAG induction, histone acetylation dynamics, and axon growth potential. Finally, DNA methylation has emerged as important epigenetic mechanism for gene regulation in neurons. TET3 is a newly characterized enzyme that catalyzes the conversion of methyl-cytosine (5mC) to hydroxymethyl-cytosine (5hmC). Our preliminary studies have identified specific upregulation of TET3, and consistently, 5hmC enrichment in the regenerating DRG neurons. Genome-wide 5hmC mapping revealed unique genomic loci with 5hmC enrichment in the regenerative condition. In Aim 3, we will determine the role of TET3 and 5hmC in RAG induction and axon growth potential by both genetic and epigenetic approaches. The outstanding strength of our proposal is an integral approach of tackling both extrinsic and intrinsic barriers to axon regeneration. Our study is expected to prioritize currently available reagents and identify novel epigenetic targets and therapeutic directions for SCI.